Korean J Hematol.  2008 Mar;43(1):19-27. 10.5045/kjh.2008.43.1.19.

Abrogation of U266 Multiple Myeloma Cell Proliferation Via Inhibition of NF-kappaB Activation by Curcumin

• Park J
• Ahn KS
• Bae EK
• Kim JH
• Jung SH
• Kim BS
• Kim DY
• Kim BK
• Lee YY
• Yoon SS

• KMID: 1485153
• DOI: http://doi.org/10.5045/kjh.2008.43.1.19

Abstract

BACKGROUND: Curcumin is a naturally occurring biologically active compound, and it has been shown to possess potent anti-inflammatory, anti-tumor and anti-oxidative properties. It is known for its anti-proliferative and proapoptotic effects in several cancer cells. Curcumin's effects on the mechanisms of cell survival and the expression of various cytokines were investigated in U266 cells and the in vivo effects of curcumin were examined using an animal model.
METHODS
Cell proliferation assay and flow cytometry were used to examine cell proliferation, along with cell cycle analysis. The protein expressions were analyzed by Western blotting and the expressed levels of cytokines were analyzed by the ELISA method.
RESULTS
Curcumin inhibited U266 cell growth in a dose-dependent and time-dependent manner. Cell cycle analysis showed an increased sub-G1 phase, a down regulated cyclinD1 expression and an induced p21 expression. Apoptosis induced a down regulated procaspase 3 expression and it induced cleaved PARP. Curcumin inhibited the IL (interleukin)-6 induced cell signal pathway via decreasing the STAT1 an 3, Erk cyclinD1 and c-myc expressions. Also, administration of 25mg/kg curcumin to a U266 animal model inhibited cancer cell engraftment in the bone marrow and it decreased the IL-6, sIL-6R and IL-8 expression levels.
CONCLUSION
Curcumin induced cell cycle arrest and apoptosis and it inhibited the IL-6 mediated signal transduction pathways in U266 cells. Similar to the in vitro results, curcumin inhibited cancer cell proliferation and the expression of cytokine in vivo.

Keyword

Curcumin; 6-aminoquinazoline; Multiple myeloma; Animal model; IL-6; sIL-6R

MeSH Terms

Animals
Apoptosis
Blotting, Western
Bone Marrow
Caspase 3
Cell Cycle
Cell Cycle Checkpoints
Cell Proliferation
Cell Survival
Curcumin
Cytokines
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Interleukin-6
Interleukin-8
Models, Animal
Multiple Myeloma
NF-kappa B
Peptides
Signal Transduction
Caspase 3
Curcumin
Cytokines
Interleukin-6
Interleukin-8
NF-kappa B
Peptides

Figure

• 

  Fig. 1 The growth of U266 cells was inhibited and activated apoptotic related protein by curcumin treatment. Curcumin treated with at various concentrations and times and curcumin (50∼75μM) reduced cell viability by 40∼60% for 72 hrs (A). U266 cell treated with indicated concentration of curcumin for 24 hrs. Apoptosis induced by exposure of 25 and 50μM concentration of curcumin (B).

• 

  Fig. 2 Sub-G1 was induced by curcumin treatment via inactivation of cyclin D1 in U266 cells. Sub-G1 phase increased after treating U266 cells with curcumin 25μM for 24 hrs (A). U266 cell treated with indicated concentration of curcumin for 24 hrs. Curcumin treatment decreased expression of cyclinD1 and CDK4. Its treatment increased expression of p21 (B).

• 

  Fig. 3 Effect of curcumin including various signal transduction inhibitors on IL-6 mediated cell signal in U266 cells. U266 cells were incubated with serum free culture media (RPMI-1640) for 24 hours. U266 cell pretreated with various signal transduction inhibitors including curcumin for 1hr and then stimulated with 5ng human recombinant IL-6 for 15 min. 30μg of whole-cell extracts were analyzed by Western blot for phosphorylated STAT1, STAT3, total STAT1, STAT3, phosphorylated Erk, and total Erk (A). Cyclin D1, c-myc and β-actin (B). Lane: 1. control; 2. 5ng/mL IL-6; 3. 25μM curcumin, 5ng/mL IL-6; 4. 20μM LY294002, 5ng/mL IL-6; 5. 10 μM PD98059, 5ng/mL IL-6; 6. 20μM SP600125, 5ng/mL IL-6; 7. 20μM AG490, 5ng/mL IL-6; 8. 10μM SB203580, 5ng/mL IL-6; 9. 2μM 6-amino-4-quinazoline, 5ng/mL IL-6.

• 

  Fig. 4 Secretion of IL-6 and sIL-6R were inhibited by curcumin treatment. U266 cell treated with indicated concentration of curcumin for 24 hrs after culture in serum free media for 24 hrs. The secretion of IL-6 (A) and sIL-6R (B) was decreased at treated with curcumin.

• 

  Fig. 5 Effect of curcumin in U266 animal model using NOD/SCID mouse. After U266 (5×105) cells were given via NOD/SCID mouse lateral tail vein, 25mg/kg was injected via intraperitoneum. 6 weeks later, development of bone lesions was examined using whole body micro CT imaging of NOD/SCID mouse. (A) Control, (B) Curcumin-injected SCID mouse.

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  Fig. 6 Secretion of IL-6 and sIL-6R were inhibited by curcumin treatment. After administration of 25mg/kg curcumin to U266 animal model for 6 weeks, IL-6, sIL-6R and IL-8 expression levels were examined using ELISA assay. The expression of IL-6 (A), sIL-6R (B) and IL-8 (C) in serum of administration of curcumin group was decreased with statically significance.

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